The present invention generally relates to differentiating and/or separating portions of a sample that are of interest from the remainder of the sample. For example, embodiments may be directed towards systems, devices, and methods for differentiating, separating, and/or sorting cells of interest from a cell sample.
In the development of drugs, or for the diagnosis and monitoring of disease, cells must be characterized, and often sorted for further analysis. A workhorse in this field is the flow cytometer. Measurements are performed on cells in liquid suspension, which flow one by one through a focused laser beam at rates up to several thousand cells per second. Light scattered and often the fluorescence emitted by the cells, or cell ‘type’-specific fluorescent-labels attached to cells is collected, filtered, the data digitized and sent to a computer for analysis. In the research lab the technology has a number of applications. These include: cell viability assays, DNA/RNA analysis, immunophenotyping, signal transduction assays, apoptosis assays, drug resistance profiling, and protein expression analysis. Clinicians have begun to use flow cytometry for cancer and HIV diagnosis and in characterizing the likelihood of organ transplant rejection.
Flow cytometry enables three important distinctions to be made by researchers and clinicians. Flow cytometry: (1) analyzes a population of cells on a cell-by-cell basis, a critical capability for those who are looking for a relatively few very specific cells among many other cell types in a sample that will enable them to study a disease state or biological process; (2) is very rapid-routine sample analysis rates can range up to 10,000 cells per second, an incredible advance over historical methods of visually examining and counting cells; and (3) has the capacity to simultaneously measure multiple characteristics/attributes of single cells. Multi-parametric analysis allows researchers and clinicians to gather more information from a single sample faster than ever before. For example, a high-end system might have 4 lasers and be capable of processing data from as many as 18 fluorophores.
An additional feature of flow cytometers is their ability to sort cells. Cell sorting with flow cytometry is known as fluorescence activated cell sorting (FACS). In FACS, cells are funneled single-file through a narrow opening that ends in a nozzle, such that droplets of fluid emerge one at a time. Each droplet may contain one or more cells. As the droplet falls, it passes through a laser (or several lasers). If the cell is labeled with a fluorescent dye that is excited by the laser light, the fluorescence signal that it subsequently emits will be ‘noted’ by detectors. The scatter of the laser light, as well as the fluorescent signal(s), tells a computer to which (pre-specified) population each droplet belongs. The droplet is collected, and the computer directs the FACS instrument to send it to the appropriate location (e.g., using electric field forces). For example, it may sort droplets into categories of no cell, cell with no fluorescent signal, cell with a green fluorescent signal, cell with a red fluorescent signal, and cell with both green and red fluorescent signals.
Flow cytometers will continue to play a major role as new molecular diagnostic and monoclonal antibody tests will facilitate existing procedures and provide a basis for additional sensitive, specific and simple assays. However, presently there exist time-consuming analyses of data, including chromosomal abnormalities, DNA content, and lymphocyte subsets that reduce the effectiveness of flow cytometry. Further, the addition of capabilities adds costs, especially with new lasers and fluorescent markers. With multiple lasers and fluorophores, costs become an important consideration, as does compensation (systematic, but arbitrary reduction in signal to account for overlapping fluorescence signals), and signal/noise issues.
While advances have been made in the art of cell and/or particle sorting and filtering from a heterogeneous sample, further improvements may be desired that provide additional differentiation parameters, sorting capabilities, improve sensitivity, sort based on additional parameters based on cell type, and may do so using cost effective methods and components.